The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of common general knowledge in the field.
Systems which require multiple or cyclic chemical reactions to produce a desired product often require careful temperature control, and reproducible and accurate control over the time the reaction is held at temperature. Such reactions include, for example, nucleic acid amplification reactions such as the polymerase chain reaction (PCR) and the ligase chain reaction (LCR).
PCR is a technique involving multiple cycles that results in the geometric amplification of certain polynucleotide sequences each time a cycle is completed. The technique of PCR is well known and is described in many books, including, PCR: A Practical Approach, M. J. McPherson, et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications by Innis, et al., Academic Press (1990), and PCR Technology: Principals and Applications for DNA Amplification H. A. Erlich, Stockton Press (1989). PCR is also described in many US patents, including U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; and 5,066,584. The content of these documents is incorporated herein by reference in their entirety.
The PCR technique typically involves the step of denaturing a polynucleotide, followed by the step of annealing at least a pair of primer oligonucleotides to the denatured polynucleotide, i.e., hybridizing the primer to the denatured polynucleotide template. After the annealing step, an enzyme with polymerase activity catalyzes synthesis of a new polynucleotide strand that incorporates the primer oligonucleotide and uses the original denatured polynucleotide as a synthesis template. This series of steps (denaturation, primer annealing, and primer extension) constitutes a PCR cycle.
As cycles are repeated, the amount of newly synthesized polynucleotide increases geometrically because the newly synthesized polynucleotides from an earlier cycle can serve as templates for synthesis in subsequent cycles. Primer oligonucleotides are typically selected in pairs that can anneal to opposite strands of a given double-stranded polynucleotide sequence so that the region between the two annealing sites is amplified.
Denaturation of DNA typically takes place at around 90 to 95° C., annealing a primer to the denatured DNA is typically performed at around 40 to 60° C., and the step of extending the annealed primers with a polymerase is typically performed at around 70 to 75° C. Therefore, during a PCR cycle the temperature of the reaction mixture must be varied, and varied many times during a multicycle PCR experiment.
In order to speed up the overall analysis time, there is a need to be able to bring the reagents to the desired temperature quickly, and for the reaction to be uniformly held at temperature for a discrete amount of time before bringing the reaction to the next temperature in the cycle. There is also a need for accurate temperature control over the reactants.
A number of thermal “cyclers” used for DNA amplification and sequencing are disclosed in the prior art in which one or more temperature controlled elements or “blocks” hold the reaction mixture, and wherein the temperature of the block is varied over time. These devices suffer the drawback that they are slow in cycling the reaction mixtures and temperature control is less than ideal. In an effort to overcome the need to cyclically raise and lower the temperature of the heating blocks, others have devised apparatus known in the art as a thermocycler. In this apparatus, multiple temperature controlled blocks are kept at different desired temperatures and a robotic arm is utilized to move reaction mixtures from block to block. Typical thermocycler systems are disclosed in U.S. Pat. Nos. 5,443,791; 5,656,493 and 6,656,724. However, as will be appreciated, these systems suffer from their own set of drawbacks. For example, they have a relatively limited throughput, they are physically large, prone to break down, expensive and require constant routine maintenance.
Various attempts have been made in the prior art to reduce the overall cycle time and/or improve temperature control, and generally address the above-mentioned disadvantages. The most common methods are non-contact and rely on hot air cycling, which is carried out by rapidly switching streams of air at the desired temperature. However the control and application of hot air is not efficient or readily controllable.
An advance over such prior art devices was first disclosed in International PCT Publication No. WO 98/49340, which teaches a thermocycler using a rotatable platform for amplification and detection of DNA fragments. Reagents are loaded into the loading wells of the rotatable platform, and upon rotation of the platform are mixed together and centrifugally displaced into the reaction wells, which are distributed about the periphery of the platform. The rotatable platform is then thermally cycled. By rotating the platform the individual reaction wells can be continuously monitored by a fixed detector. Thermal cycling of the platform is effected with conventional heating methods, such as by use of a heating element to heat a stream of air which is directed at the platform. The disadvantage of heating the entire platform with hot air is that the surrounding structures in the device will also become heated, which will need to be cooled in the cooling phase of the cycle otherwise they will continue to radiate heat and will affect the temperature of the reactions occurring in the reaction wells. Heating and cooling parts of the device other than the platform itself is inefficient, and temperature control using a stream of heated air is less than ideal. Also, it is difficult to measure the temperature of the reaction mixture, which therefore needs to be estimated. Because the temperature differential between air and the reaction mixture is very large, the estimated reaction temperature is subject to very large errors, meaning that there is poor temperature control.
Other methods of heating disclosed in WO 98/49340 comprise directing a narrow beam of IR light or microwave energy at a portion of the platform and then rotating the platform through or past the beam. In this way, each portion of the platform is effectively “pulsed” with energy, and as such only a small portion of the platform is heated at any one time. This can lead to a thermal differential across the platform. In WO 98/49340, the platform is cooled by exposing the rotatable platform to a stream of coolant fluid, such as ambient air, which is optionally chilled. In summary, the heating methods employ non-coherent and non-focused sources of electromagnetic energy, which require high power for the reaction wells to reach the required temperature. Additionally, heating the reactions via these conventional means can take minutes to reach the predetermined set-point temperature.
In light of forgoing discussion, it is a preferred object of the present invention to develop a non-contact real-time thermocycler which has improved thermal cycling speed and which therefore reduces the overall cycle time.
It is an object of the present invention to overcome or ameliorate one or more of the disadvantages of the prior art, or at least to provide a useful alternative.